1. Field of the Invention
The present invention relates to a method of and apparatus for immunologically analyzing an object of measurement in the field of clinical testing, biochemical sample measurement, quality control of drugs and the like.
2. Description of the Background Art
Immunological analysis methods employing antigen-antibody reaction include fluorescent immunoassay and luminous immunoassay. In any one of these methods, an antigen which is labeled with a fluorescent material or a chemiluminescent material is reacted to an antibody with an antigen which is an object of measurement in competition, to form an immune complex which is a molecular complex by the antigen-antibody reaction for measurement fluorescence or luminescence from the label, thereby quantitatively analyzing the target material.
On the other hand, a method of adding an antigen of an antibody of an object of measurement to an antibody or an antigen and measuring absorption or scattering of light by an immune complex which is formed by antigen-antibody reaction thereby making quantitative analysis is known as an optical measuring method utilizing neither fluorescence nor luminescence. Quantitative analysis methods utilizing light scattering phenomenona include turbidimetry and nephelometry. The former is adapted to measure transmitted light which is attenuated by absorption and scattering, while the latter is adapted to measure scattering light intensity. The method by light scattering is adapted to measure Rayleigh scattering or Mie scattering, in response to sizes of particles as measured.
A fluorescent immunoassay apparatus belonging to fluorescent immunoassay is proposed as an immunoassay apparatus employing a total reflection cell (refer to Japanese Patent Laying-Open Gazette No. 5-203574 (1993)). In this fluorescent immunoassay, an antibody is fixed to a surface of a total reflection prism, an antigen contained in a sample is bonded to the antibody by antigen-antibody reaction, and an antibody which is labelled with a fluorescent material is further bonded to the antigen by antigen-antibody reaction. Thereafter B-F separation is performed to remove an unreacted antibody which is labelled with the fluorescent material, and then an excitation beam is introduced into the total reflection prism to excite the labelled antigen-antibody immune complex which is constrained on the surface of the total reflection prism, for measuring fluorescence generated from the fluorescent material.
The method utilizing light scattering, which is homogeneous immunoassay, is a simple method requiring neither B-F separation nor washing. However, the method utilizing Rayleigh scattering or Mie scattering has problems of low detection sensitivity and low measuring accuracy in relation to a low concentration material.
On the other hand, the fluorescent or luminescent immunoassay requires complicated chemical treatment for labelling an antigen or an antibody with a fluorescent or chemiluminescent material. Further, most thereof is heterogeneous immunoassay, which inevitably requires B-F separation for separating an immune complex (B) making antigen-antibody reaction from an antigen or antibody (F) making no antigen-antibody reaction and washing through a large number of analytical steps.